# Patent

## Title
Method for prediction of sensitivity to 5-fluorouracil-type anticancer agent

## Abstract

A factor affecting sensitivities to anticancer agents is analyzed and the validity of the factor is demonstrated.
The present invention provides a method for predicting sensitivities to 5-fluorouracil-based anticancer agents using the copy number of dihydropyrimidine dehydrogenase gene as an indicator. Also disclosed are a kit and primers for use in predicting sensitivities to 5-fluorouracil-based anticancer agents.

## Background
TECHNICAL FIELD
The present invention relates to a method for predicting sensitivities to 5-fluorouracil-based anticancer agents.
BACKGROUND ART
DNA copy number alterations are one of the many causes which affect gene expression alterations. In recent years, comprehensive search methods using such as array CGH have been employed in the analysis of DNA copy numbers, and such methods have been gradually demonstrated to be useful. An epoch-making article which identified the causative gene for melanoma drug resistance from analyses of NCI60 screening panel cell lines with 100K SNP arrays (Affymetrix) was published in Nature in July last year (Non-Patent Document 1). The raw data contained in the article are disclosed in NCI database and invite re-analysis by the third party. On the other hand, no report has been made about correlation between abnormalities in DNA copy numbers and the efficacies of existing anticancer agents.
To date, as a method for predicting sensitivities to 5-fluorouracil-based anticancer agents, determination of the enzyme activity of dihydropyrimidine dehydrogenase prepared from a cancer cell line or cancer tissue, determination of the quantity thereof by ELISA, or determination of its mRNA expression level has been attempted (Non-Patent Documents 2 and 3, Patent Document 1). However, enzyme or mRNA is easily degraded when fresh tissues from surgical operation are stored. Yet, the degree of degradation varies depending on the laboratory. Thus, it has been difficult to use the enzyme activity, quantity or mRNA expression level as a common indicator for predicting sensitivity. Furthermore, a large amount of tissue is required for determination of enzyme activity, and the amount of sample obtainable from biopsy or the like was insufficient to enable such determination. Besides, determination of mRNA expression levels using paraffin-embedded samples which are widely used in histopathology is difficult in terms of maneuver.

    [Non-Patent Document 1]

Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma: Garraway-L A, et al. Nature (2005) vol. 436, 7 July, pp 117-121

    [Non-Patent Document 2]

Dihydropyrimidine dehydrogenase activity in human peripheral blood mononuclear cells and liver; population characteristics, newly identified deficient patients, and clinical implication in 5-fluorouracil chemotherapy: Lu Z, Zhang R, Diasio R B. Cancer Res. 1993 Nov. 15; 53(22):5433-8

    [Non-Patent Document 3]

A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil: Beck A, Etienne M C, Cheradame S, Fischel J L, Formento P, Renee N, Milano G. Eur J Cancer. 1994; 30A(10): 1517-22

    [Patent Document 1]

Japanese Unexamined Patent Publication (PCT) No. 2005-508603



DISCLOSURE OF THE INVENTION
Problem for Solution by the Invention
It is an object of the present invention to analyze a factor which affects sensitivities to anticancer agents and to demonstrate the utility of the factor.
Means to Solve the Problem
The present inventors have comprehensively analyzed the data used in the above-described articles and the drug sensitivity data, gene expression data and karyotype data accumulated enormously in NCI database (data mining) to thereby identify an alteration in DNA copy number which affects sensitivities to existing anticancer agents (in Examples herein, 5-FU-based anticancer agents), the alteration having been unknown to date. The present inventors have also examined whether this finding can be generalized or not using 31 human cancer-derived xenografts (not overlapping the cell lines in the NCI screening panel) obtained through subcutaneous transplantation into nude mice.
The present invention relates to the following inventions.
(1) A method of predicting the sensitivity of a cancer cell to a 5-fluorouracil-based anticancer agent using the copy number of dihydropyrimidine dehydrogenase gene as an indicator.
(2) The method of (1), comprising determining the copy number of dihydropyrimidine dehydrogenase gene in a cancer cell derived from a subject, wherein the subject's sensitivity to the 5-fluorouracil-based anticancer agent is predicted to be high when the resultant copy number is 2 or less and the subject's sensitivity to the 5-fluorouracil-based anticancer agent is predicted to be low when the resultant copy number is more than 2.

(3) The method of (1) or (2), wherein the copy number of dihydropyrimidine dehydrogenase gene is determined by at least one method selected from the group consisting of PCR method, FISH method, array CGH method, DNA microarray method and Southern hybridization method.

(4) The method of any one of (1) to (3), wherein the 5-fluorouracil-based anticancer agent comprises at least one component selected from the group consisting of 5-fluorouracil, tegafur, 5′-deoxy-5-fluorouracil and capecitabine.

(5) A kit for predicting sensitivities to 5-fluorouracil-based anticancer agents, comprising a reagent for determining the copy number of dihydropyrimidine dehydrogenase gene.

(6) The kit according of (5), wherein the reagent for determining the copy number of dihydropyrimidine dehydrogenase gene is oligonucleotide primers capable of specifically amplifying the whole or a part of dihydropyrimidine dehydrogenase gene or an oligo- or poly-nucleotide probe capable of specifically hybridizing to dihydropyrimidine dehydrogenase gene.

(7) A pair of primers composed of an oligonucleotide consisting of the DNA sequence as shown in SEQ ID NO: 5 and an oligonucleotide consisting of the DNA sequence as shown in SEQ ID NO: 6

Effect of the Invention
According to the present invention, it has been found that alteration in the copy number of dihydropyrimidine dehydrogenase gene affects the sensitivity of cancer cells to 5-fluorouracil-based anticancer agents. The present invention is applicable to prediction of subjects' sensitivities to 5-fluorouracil-based anticancer agents.
The present specification encompasses the contents described in the specification and/or drawings of Japanese Patent Application No. 2006-16367 based on which the present patent application claims priority.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a histogram showing the distribution of DPYD copy numbers in 31 xenografts. The vertical axis represents frequency and the horizontal axis represents DPYD copy number.
FIG. 2 is a heat map obtained by normalizing the data on the DPYD copy numbers and the sensitivities to FU-based anticancer agents (UFT, TS-1, capecitabine and 5′-DFUR) of 31 xenografts and then clustering with Ward's method. The results are shown in grey scale images. The higher the copy number and the sensitivity are, the darker the color is.
FIG. 3 shows box-and-whisker plots obtained by classifying 31 xenografts with DPYD copy numbers (vertical axis) and sensitivities to the individual drugs indicated below (horizontal axis). Specifically, 31 xenografts were classified into the following three groups based on their sensitivities to 5-FU-based anticancer agents (UFT, TS-1, capecitabine and 5′-DFUR): (S) high sensitivity strains within the 25th percentile, (R) low sensitivity strains which are the 75th percentile or below, and (N) intermediate strains other than the above two groups. A round robin test considering multiplicity was performed by Tukey-Kramer HSD test. The results are shown as P values.
FIG. 4 is a graph plotting correlation between the DPYD copy numbers and DPYD mRNA expression levels in 31 xenografts. The vertical axis represents DPYD copy number and the horizontal axis represents mRNA expression level. Correlation coefficient and significance were obtained using Speaman rank correlation.
FIG. 5 is a graph plotting correlation between the DPYD copy numbers and DPYD enzyme activity levels in 31 xenografts. The vertical axis represents DPYD copy number and the horizontal axis represents DPYD enzyme activity level. Correlation coefficient and significance were obtained using Speaman rank correlation.
FIG. 6 shows verification of a DNA probe using chromosome samples (mitotic phase). Arrow marks indicate a DPD gene (1p21) probe.
FIG. 7 shows the results of FISH analysis using paraffin samples (PAN-4).
FIG. 8 shows the results of FISH analysis using paraffin samples (Lu-130).



BEST MODE FOR CARRYING OUT THE INVENTION
Hereinbelow, the embodiments of the present invention will be described in detail.
The present invention provides a method of predicting sensitivities to 5-fluorouracil-based anticancer agents (hereinafter, sometimes referred to as “5-FU”) using as an indicator the copy number of dihydropyrimidine dehydrogenase gene (hereinafter, sometimes referred to “DPYD”) (i.e., the number of the gene contained in one cell).
The term “dihydropyrimidine dehydrogenase” used herein refers to an enzyme with a molecular weight of about 111 kDa (EC 1.3.1.2) which catalyzes antidotal degradation of 5-fluorouracil. This enzyme acts as a rate-limiting enzyme in the pyrimidine degradation pathway. It is known that the activity of this enzyme is high mainly in the liver. Genetic sequence information for this enzyme is registered at the NCBI web site (http://www.ncbi.nlm.nih.gov/) as RefSeq ID: NM—00110 (human) and RefSeq ID: NM—170778 (mouse). The amino acid sequence information thereof is registered in the same database as NP—000101 (human) and NP—740748 (mouse). The nucleotide sequence and amino acid sequence for human-derived dihydropyrimidine dehydrogenase are described in Diasio, R. B. et al., J. Clin. Invest. 81(1), 47-51 (1988). The nucleotide sequence and amino acid sequence for mouse-derived dihydropyrimidine dehydrogenase are described in Porsin, B. et al, Eur. J. Cancer 39 (6), 822-828 (2003).
In one embodiment of the present invention, it is possible to determine the copy number of dihydropyrimidine dehydrogenase gene in a cancer cell derived from a subject and to predict that the subject's sensitivity to 5-fluorouracil-based anticancer agents is high when the copy number is 2 or less and that the subject's sensitivity to 5-fluorouracil-based anticancer agents is low when the copy number is more than 2.
In the present invention, when a cancer cell is judged “highly sensitive” to an anticancer agent, therapeutic efficacies (such as antitumor effect, survival benefit, etc.) resulting from the administration of the anticancer agent are predicted to be high. When a cancer cell is judged “lowly sensitive” to an anticancer agent, therapeutic efficacies are predicted to be low. Therefore, the present invention also includes a method of predicting the therapeutic efficacy of a 5-fluorouracil-based anticancer agent on a cancer using as an indicator the copy number of dihydropyrimidine dehydrogenase gene. More specifically, the present invention also includes a method of predicting the therapeutic efficacy of a 5-fluorouracil-based anticancer agent on a cancer cell using the copy number of dihydropyrimidine dehydrogenase gene as an indicator, comprising determining the copy number of dihydropyrimidine dehydrogenase gene in a cancer cell derived from a subject, wherein the therapeutic efficacy on the subject by the 5-fluorouracil-based anticancer agent is predicted to be high when the resultant copy number is 2 or less and the therapeutic efficacy on the subject by the 5-fluorouracil-based anticancer agent is predicted to be low when the resultant copy number is more than 2.
In the method of the present invention, not only human but also a mammal such as pig, monkey, chimpanzee, dog, cattle, rabbit, rat or mouse may be used as the subject.
For determination of the copy number of dihydropyrimidine dehydrogenase gene in a cancer cell derived from a subject, materials containing cancer tissue or cancer cells (such as biopsy samples, removed organs, paraffin-embedded tissue samples, blood, spinal fluid, lymph, saliva, gastric juice, pancreatic juice, duodenal fluid, intestinal fluid, stool, etc.) or cultured cells or tissues obtainable therefrom may be used.
Specific types of cancer include, but are not limited to, gastric cancer, large bowel cancer, breast cancer, lung cancer, pancreatic cancer, liver cancer, ovarian cancer, prostate cancer, head and neck cancer, malignant lymphoma, leukemia, brain tumor, uterine cancer and bladder cancer.
The copy number of dihydropyrimidine dehydrogenase gene may be determined by PCR method, FISH method, array CGH method, DNA microarray method, Southern hybridization method, or the like.
PCR Method
When the copy number of dihydropyrimidine dehydrogenase gene is determined by PCR method, the following procedures may be used.
Briefly, PCR is performed using as a template the genomic DNA of a cancer cell prepared by phenol chloroform method, centrifuge column method, magnetic beads method or the like. The PCR may be performed using QIAGEN QuantiTect SYBR Green PCR kit and ABI7300 sequence detector under the PCR conditions described below: 1 cycle of 94° C., 15 min and 45 cycles of 94° C., 20 sec; 56° C., 20 sec; and 70° C., 30 sec. However, these conditions may be appropriately changed as long as the experiment can be reproduced. The invention practiced under such changed conditions is also included within the scope of the present invention. The analytical value of the target DNA may be obtained based on the PCR cycle number (Ct value) at which the PCR product reached any threshold value. Further, the gene copy number may be corrected with an analytical value obtained using a reference sequence which varies little in copy number between cancer cells and normal cells. When the copy number of dihydropyrimidine dehydrogenase gene is determined by PCR method, oligonucleotide primers may be used which are capable of specifically amplifying the whole or a part of dihydropyrimidine dehydrogenase gene. These oligonucleotide primers may be primers capable of hybridizing to a region of dihydropyrimidine dehydrogenase gene having a nucleotide sequence which may be present in cancer cells but is not found in other genomic DNA. The primer sizes are preferably about 17-25 bases. Preferably, the Tm values are set uniformly for the forward primer and the reverse primer at about 55-65° C., which is apt to produce good results. A pair of primers which have less complementarity to one another are selected so that the two primers do not anneal to one another. In order to prevent particularly the decrease in amplification efficiency due to formation of primer dimers, the primers are designed so that their 3′ end sequences are not complementary to one another in 3 or more consecutive bases. Further, in order to avoid formation of secondary structures within primers, the primers are designed so that they do not contain auto-complementary sequences of 4 or more bases. GC content is set at about 40-60% so that partial GC- or AT-rich sequences are avoided. For achieving stable binding of the 3′ ends of primers to the template DNA, the 3′ terminal sequences of the primers should not be AT-rich or GC-rich. It should be noted that when the 3′ terminal sequence of a primer is GC-rich, non-specific product are easily produced. With respect to the Tm value, the following points should be noted. The “Tm value” refers to the temperature at which 50% of double-stranded DNA is dissociated into single-stranded DNA (melting temperature). In order for primers to anneal to the template DNA to thereby start elongation, the annealing temperature must be set below the primer Tm value. However, excessive lowering of the temperature causes non-specific annealing to thereby decrease specific amplification efficiency. When there are a plurality of candidate primer pairs, usually, a pair with a high Tm value is selected in order to enhance specificity. When two primers making a pair have respective Tm values different from each other, the annealing temperature is tentatively set considering the lower Tm value. When primers have been designed taking into account of the above-described points, good results are obtained with annealing temperatures of 55-65° C. empirically, However, when no PCR product is obtained, PCR should be performed at a lowered temperature. When non-specifically amplified products are obtained, PCR should be performed at a raised temperature.
One example of the nucleotide sequences for a primer pair used in PCR method is given below.











Forward primer:





5′-CGGCCCTAGTCTGCCTGTT-3′
(SEQ ID NO: 5)



 



Reverse primer:



5′-GAGTCTGCCAGTGACAAACCCT-3′
(SEQ ID NO: 6)






As a reference genomic sequence, LINE-1 (a sequence occurring abundantly on the genome whose copy number is believed to be almost the same between cancer cells and normal cells) may be used.











Forward primer:





5′-AAAGCCGCTCAACTACATGG-3′
(SEQ ID NO: 7)



 



Reverse primer:



5′-TGCTTTGAATGCGTCCCAGAG-3′
(SEQ ID NO: 8)






FISH Method

When the copy number of dihydropyrimidine dehydrogenase gene is determined by FISH method, an oligo- or poly-nucleotide probe capable of specifically hybridizing to dihydropyrimidine dehydrogenase gene may be used. This oligo- or poly-nucleotide probe may be a probe capable of hybridizing to a region of dihydropyrimidine dehydrogenase gene having a nucleotide sequence which may be present in cancer cells but is not found in other genomic DNA.
As the probe for FISH, a DNA fragment, a PCR product, a cDNA, a PAC clone or a BAC clone (each of which has a sequence of interest) may be used. In situ hybridization method has been developed as a method for examining the presence/absence and distribution of a specific DNA or RNA (nucleic acid) in cells or tissues. This method utilizes the nature of a nucleic acid probe having a nucleotide sequence complementary to a specific nucleic acid in cells, i.e., such a probe specifically forms a complex (hybridization). When such probes are labeled with radioisotopes (RIs) or antigenic substances (haptens) in advance, the site of hybridization becomes discriminable. Conventionally, RIs have been used for labeling probes. Recently, however, fluorescence labeling methods or detection methods utilizing non-radioactive substances, e.g., haptens such as biotin or digoxigenin, have been developed. Among them, fluorescence in situ hybridization method called FISH has been developed (Tohyama (ed.). Experimental Medicine Special Issue, “In situ Identification of Proteins and Nucleic Acid Molecules”).
Procedures for FISH method will be illustrated below. Chromosome samples may be smears on slide glass prepared from cultured cells of an isolated cancer. Alternatively, chromosome samples may be slide samples sliced from formalin-fixed, paraffin-embedded cancer-containing tissue blocks. After hardening for prevention of falling off from the slide glass during hybridization process, chromosome sample slides are denatured by formamide treatment. In a FISH method where biotin is used as a labeling substance, a probe DNA is labeled with biotin-dUTP (or biotin-dATP) and then the DNA is thermally denatured. Subsequently, the DNA probe is subjected to hybridization to single-stranded DNA. The resultant double-stranded DNA composed of the biotin-labeled DNA and the genomic DNA is washed with a washing solution (major component is SSC buffer) and treated with avidin-FITC solution which has a high affinity to biotin. Subsequently, the double-stranded DNA is washed with a series of SSC buffer, followed by addition of an antibleaching agent thereto drop wise. The resultant DNA is covered with a cover glass, observed under a fluorescence microscopy and photographed.
Array CGH Method
CGH (comparative genomic hybridization) method is an analytical method for specifying a chromosome in which an abnormality is occurring with a fluorescent dye and is one type of FISH method. Conventional CGH method was low in dissolution capacity and it was hard to identify a target gene with the resultant genomic abnormality data. This time, the target genomic region in which copy number abnormalities are to be detected is definite. Therefore, it is possible to detect copy number abnormalities by the CGH method described below. Briefly, DNA is extracted from a cancer cell and a normal cell (used as a control). The cancer cell-derived DNA is labeled with a green fluorescent dye (FITC) and the normal cell-derived DNA is labeled with a red fluorescent dye (Texas red). A mixed solution consisting of equal amounts of the two labeled DNAs is prepared and subjected to hybridization. In conventional CGH method, as samples to be hybridized, chromosome sample slides prepared by taking blood samples from human, culturing them, terminating the cell division at metaphase, and smearing cells on slide glass with the cell membrane being exposed were used. However, by performing CGH using a slide glass with a large number of cloned DNA fragments set in array (array CGH), it is possible to quantitatively determine the copy number of cancer DNA in a region corresponding to a DNA fragment in the array, based on the intensity ratio between fluorescence signals from labeled cancer cell- and normal cell-derived DNAs. Examples of DNAs to be set in array on a slide glass include, but are not limited to, BAC clones which are 100 kb human genomic fragments cloned or products obtained by amplifying cloned genomic fragments by DOP-PR with BAC as a template.
Microarray Method
It is possible to detect alterations in copy number in a specific region in the genome using commercial DNA microarrays (oligonucleotide arrays or cDNA microarrays) for SNP detection in the same manner as in array CGH method. For DNA preparation method and labeling method, standard protocols provided in commercial microarrays may be used.
Southern Hybridization Method
Southern hybridization method is a classic method for detecting a DNA of interest utilizing complementarity in nucleic acid. Briefly, genomic DNAs prepared from a cancer cell and a normal cell are treated with appropriate restriction enzymes and electrophoresed on agarose gel to fractionate depending on the DNA size. After denaturation into single-stranded DNA by alkali denaturation, the resultant DNA is transferred onto a filter (such as nitrocellulose) and fixed. A probe which is a DNA fragment comprising a target sequence labeled with a radioisotope or the like is reacted with the filter. Thus, it is possible to detect alterations in copy number of a genetic region of interest.
The copy number of dihydropyrimidine dehydrogenase gene may be determined by any of the above-described methods. When the copy number of dihydropyrimidine dehydrogenase gene is 2 or less, it is possible to predict that the subject's sensitivity to 5-fluorouracil-based anticancer agents is high. When the copy number of dihydropyrimidine dehydrogenase gene is more than 2, it is possible to predict that the subject's sensitivity to 5-fluorouracil-based anticancer agents is low. If it is possible to predict the sensitivity of a cancer patient to 5-fluorouracil-based anticancer agents, selection of appropriate drugs or avoidance of unnecessary drug administration becomes possible in cancer treatment. Thus, it becomes possible to make an appropriate administration plan or to change to an appropriate administration plan.
The method of the present invention has the following advantages. By using as a material DNA which is unlikely to undergo degradation and by using a universal indicator (gene copy number), the method of the present invention enables prediction of sensitivities to 5-FU-based anticancer agents with DPYD as a standardized indicator among various laboratories. Further, by using a wide range of samples including paraffin-embedded samples as a material, prediction of sensitivities can be practiced easily.
Specific examples of 5-fluorouracil-based anticancer agents include single drugs and combined drugs such as 5-fluorouracil, tegafur, 5′-deoxy-5-fluorouracil, capecitabine, UFT (combined drug comprising tegafur and uracil at a molar ratio of 1:4), TS-1 (combined drug comprising tegafur, gimeracil and oteracil potassium at a molar ratio of 1:0.4:1), carmofur, yamaful and sunfural. Among all, anticancer agents comprising at least one component selected from the group consisting of 5-fluorouracil, tegafur, 5′-deoxy-5-fluorouracil and capecitabine are preferred.
The present invention also provides a kit for predicting sensitivities to 5-fluorouracil-based anticancer agents, comprising a reagent for determining the copy number of dihydropyrimidine dehydrogenase gene.
As the reagent for determining the copy number of dihydropyrimidine dehydrogenase gene, oligonucleotide primers capable of specifically amplifying the whole or a part of dihydropyrimidine dehydrogenase gene, an oligo- or poly-nucleotide probe capable of specifically hybridizing to dihydropyrimidine dehydrogenase gene, or the like may be given. These primers and probes are as described above. The kit of the present invention may further comprise other reagents, buffers and handling manuals necessary for determination of the copy number of dihydropyrimidine dehydrogenase gene by PCR method, FISH method, array CGH method, DNA microarray method, Southern hybridization method or the like. It is preferred that the handling manual describe criteria for predicting sensitivities to 5-fluorouracil-based anti-cancer agents in addition to how to use the kit.
Further, the present invention provides a pair of primers composed of an oligonucleotide consisting of the DNA sequence as shown in SEQ ID NO: 5 and an oligonucleotide consisting of the DNA sequence as shown in SEQ ID NO: 6. By using the pair of primers of the present invention, it is possible to determine by PCR method the copy number of dihydropyrimidine dehydrogenase gene in a cancer cell derived from a subject and to predict the subject's sensitivity to 5-fluorouracil-based anti-cancer agents based on the results of the determination.
EXAMPLES
Hereinbelow, the present invention will be described in more detail with reference to the following Examples. However, the present invention is not limited to these Examples.
Example 1
Examination in Human Tumor Strains Subcutaneously Transplanted into Nude Mice
As a result of deep data-mining of the NCI60 profiling data, the present inventors have found abnormalities in the copy number of a gene that affect sensitivities to 5-FU. As an independent data set for examining whether this finding can be generalized or not, in vivo human tumor strains (xenografts: various organ cancer-derived 31 strains subcultured through subcutaneous transplantation into nude mice) were used. Particulars of the 31 xenografts are 6 gastric cancer strains (AZ-521, SC-2, ST-40, 4-1ST, SC-4 and OCUM-2MD3), 6 large bowel cancer strains (KM12C, HCT-15, KM20C, COL-1, KM12C/FU and CO-3), 6 breast cancer strains (MC-5, H-31, MC-2, MX-1, MDA-MB-435SHM and MDA-MD-231), 7 lung cancer strains (GT3TKB, LC-11, Lu-99, LX-1, LC-6, Lu-134 and Lu-130) and 6 pancreatic cancer strains (PAN-3, PAN-4, PAN-12, H-48, MIAPaCa-2 and BxPC-3). The suppliers of these cancer strains are as described below. KM12C and KM20C were provided by Dr. Morikawa (National Cancer Institute). KM12C/FU is a strain in which FU resistance was induced in vivo. MDA-MB-435SHM is a high lung metastasis strain created by transplanting a cell line purchased from ATCC into the mammary gland of SCID mouse in vivo. LX-1 and MX-1 were provided by Dr. Inoue (Cancer Chemotherapy Center of JFCR); H-31 and H48 were provided by Dr. Taguchi (Osaka University). AZ-521 and MDA-MB-231 were purchased from Human Science Research Resource Bank and ATCC, respectively HCT-15 and BxPc-3 were purchased from Dainippon Pharmaceutical. The other strains were purchased from Central Institute for Experimental Animals. These tumor sections and cell suspensions were subcutaneously transplanted into nude mice (Male BALB/c-nu/nu nude mice; 5-week old; body weights 18 to 20 g) (purchased from CLEA Japan, Inc.). When the tumor volume (0.5×length×width2) reached 100-300 mm3, 5 mice were allocated to each test group by stratified random allocation using tumor volume as an indicator (day 0). It was confirmed that there was no significant difference in tumor volume between groups. 5-FU-based anticancer agents of UFT, TS-1, 5′-DFUR and Capecitabine were administered orally at the optimal dose (MTD) of each agent for two weeks to thereby determine antitumor effects. The antitumor effect was obtained by examining to what extent the tumor weight of drug-administered mice was inhibited at day 15 as compared to the weight of subcutaneous tumor in control mice which received no drug treatment. Briefly, tumor growth inhibition ratio IR % was calculated by the following formulas. Every experiment was performed in accordance with the ethic guideline concerning animal experiments.

RTV=(tumor weight at day 15 of treatment)/(tumor weight before treatment)

IR %=(1−mean RTV in treatment group/mean RTV in control group)×100

In order to obtain the copy number of DPYD gene, primers with the following sequences were designed.











Forward primer:





5′-CGGCCCTAGTCTGCCTGTT-3′
(SEQ ID NO: 5)



 



Reverse primer:



5′-GAGTCTGCCAGTGACAAACCCT-3′
(SEQ ID NO: 6)






As a reference genomic sequence, LINE-1 (a sequence occurring abundantly on the genome whose copy number is believed to be almost the same between cancer cells and normal cells) may be used.











Forward primer:





5′-AAAGCCGCTCAACTACATGG-3′
(SEQ ID NO: 7)



 



Reverse primer:



5′-TGCTTTGAATGCGTCCCAGAG-3′
(SEQ ID NO: 8)






The gDNA of each xenograft was prepared using QIAAMP DNA mini kit (Qiagen Inc., Valencia, Calif.) according to the manufacturer's instructions.
PCR reaction was performed using QIAGEN QuantiTect SYBR Green PCR kit and ABI7300 sequence detector under the PCR conditions described below: 1 cycle of 94° C., 15 min and 45 cycles of 94° C., 20 sec; 56° C., 20 sec; and 70° C., 30 sec.
The analytical value of the target DNA was calculated from a regression equation created by preparing calibration curves based on the PCR cycle number (Ct value) at which the PCR product reached any threshold value. Further, the gene copy number was calculated by the following formula.

(T(DPYD)/T(LINE-1))/(C(DPYD)/C(LINE-1))×2

(wherein T is the analytical value of DPYD or LINE-1 in the target tumor DNA; and C is the analytical value of DPYD or LINE-1 in the human (male) normal tissue-derived genomic DNA used as a control.)

The total RNA from each xenograft was prepared from a tissue with a wet weight of 20-30 mg using RNeasy (Qiagen Inc., Valencia, Calif.) according to the manufacturer's instructions.
Quantitative determination of DPYD mRNA was performed by TaqMan real-time reverse transcription PCR using TaqMan EZ RT-PCR kit and ABI Prism sequence detector. Standard curves were prepared with serial dilutions of the total RNA from MiaPaCa-2. As internal standards, GAPD and ACTB were quantitatively determined at the same time. The quantity of DPYD mRNA was corrected using the geometric means of both GAPD and ACTB.
Sequences for PCR primers and TaqMan probes are described below.









DPYD




5′-AATGATTCGAAGAGCTTTTGAAGC-3′ (forward primer)
(SEQ ID NO: 9)


 


5′-GTTCCCCGGATGATTCTGG-3′ (reverse primer)
(SEQ ID NO: 10)


 


5′-TGCCCTCACCAAAACTTTCTCTCTTGATAAGGA-3′ (TaqMan probe),
(SEQ ID NO: 11)


PCR amplification size: 108 bp


 


ACTB


5′-TCACCCACACTGTGCCCATCTACGA-3′ (forward primer)
(SEQ ID NO: 12)


 


5′-CAGCGGAACCGCTCATTGCCAATGG-3′ (reverse primer)
(SEQ ID NO: 13)


 


5′-ATGCCCTCCCCCATGCCATCCTGCGT-3′ (TaqMan probe),
(SEQ ID NO: 14)


PCR amplification size: 295 bp


 


GAPD


5′-GAAGGTGAAGGTCGGAGTC-3′ (forward primer)
(SEQ ID NO: 15)


 


5′-GAAGATGGTGATGGGATTTC-3′ (reverse primer)
(SEQ ID NO: 16)


 


5′-CAAGCTTCCCGTTCTCAGCC-3′ (TaqMan probe),
(SEQ ID NO: 17)


PCR amplification size: 226 bp






PCR reaction conditions were 1 cycle of 50° C., 2 min; 1 cycle of 60° C., 30 min; 1 cycle of 95° C., 5 min; and 45 cycles of 94° C., 20 sec and 60° C., 1 min.
Enzyme Activity of Dihydropyrimidine Dehydrogenase
The enzyme activity of dihydropyrimidine dehydrogenase was determined by the following method. Briefly, 4 volumes of homogenization buffer [20 mM potassium phosphate (pH 8.0) containing 1 mM EDTA and 1 mM β-mercaptoethanol] was added to a tumor tissue, and the resultant mixture was sonicated. The homogenate was centrifuged at 105,000 g for 1 hour at 4° C. and the resultant supernatant was collected. Enzyme reaction was performed with the following composition; 10 mM potassium phosphate (pH 8.0), 0.5 mM EDTA, 0.5 mM β-mercaptoethanol, 2 mM dithiothreitol, 5 mM MgCl2 and 20 μM [6-14C] 5-FU.
100 μM NADPH and 25 μl of cell supernatant (reaction volume; 50 μl) were incubated at 37° C. for 30 min. After the reaction, 5 μl of the reaction solution was subjected to thin layer chromatography (Silica gel 60F254; Merck, Germany) and developed with a mixed solution of ethanol and 1 M ammonium acetate (5:1, v/v). Dihydropyrimidine dehydrogenase activity was determined by measuring the sum of dihydrofluorouracil and 2-fluoro-β-alanine dissociated from [6-14C] 5-FU with a liquid scintillation counter. Drugs used: UFT, TS-1 and capecitabine were synthesized by Taiho Pharmaceutical Co., Ltd. 5′-DFUR was purchased from Roche Japan. [6-14C]-5-FU (1.85 GBq/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, Calif., USA).
Experimental Results
In order to examine whether the novel finding in NCI60 cells (i.e., correlation between the copy number of DPYD gene and sensitivity to 5-FU) is a phenomenon that may occur generally or not, similar examination was performed in 31 human cancer-derived xenografts obtained by subcutaneous transplantation into nude mice which do not overlap NCI60 cell strains (except for 1 xenograft).
The copy number of DPYD gene in the 31 xenografts distributed widely from 0.78 to 4.4, though the copy number was around 2 in many xenografts (Table 1, FIG. 1).
Correlation between the copy number of DPYD gene in the 31 xenografts and the efficacy of anticancer agents (UFT, TS-1, 5′-DFUR and capecitabine) therein was examined. FIG. 2 shows a heat map of the copy number and in vivo efficacy data.
As a result of unsupervised-2-way clustering, the copy number and the in vivo efficacy showed mutually contradictory patterns. Then, the inventors ranked the 31 xenografts according to their sensitivities to drugs. Xenografts within the highest 25th percentile were classified into high sensitivity group; 75th percentile or below were classified into low sensitivity group; and the other xenografts were classified into intermediate group. In each of these three groups, Tukey-Kramer HSD test was performed against the copy number of DPYD gene (FIG. 3). As a result, a significant alteration in copy number was recognized between the high sensitivity group and the low sensitivity group with respect to UFT, 5′-DFUR and capecitabine. That is, the copy number of DPYD gene in the low sensitivity group was significantly higher than the copy number in the high sensitivity group (P<0.05). Although no significant difference was observed with respect to TS-1, a significance tendency was recognized.
In NCI60 screening cell strains, a significant positive correlation was observed between the copy number of DPYD gene and the expression of DPYD mRNA. In order to examine whether this finding is also applicable to the 31 xenografts or not, correlation between the copy number of DPYD gene and the expression of DPYD mRNA was investigated in the xenografts. The results revealed a significant correlation between them (Spearman Rho=0.36, p=0.046) (FIG. 4). When correlation between the copy number and the DPYD enzyme activity was examined, a positive correlation tendency (P<0.07) was recognized (FIG. 5)











TABLE 1







Xeno_Name
Copy_Number
SE









Co-3
2.071
0.571



HCT-15
2.410
0.428



KM12C
1.226
0.132



KM20C
2.080
0.313



LC-11
1.326
0.178



Lu-99
0.772
0.263



LX-1
1.846
0.354



MC-2
1.668
0.219



MX-1
2.519
0.587



PAN-12
1.895
0.238



PAN-3
2.009
0.372



SC-2
1.935
0.298



ST-40
1.159
0.163



4-1ST
1.526
0.425



SC-4
1.934
0.492



COL-1
2.132
0.359



MDA435
4.449
2.690



BxPC-3
2.571
0.379



Lu-130
3.060
0.670



KM12FU
1.086
0.171



LC-6
2.101
0.402



MIAPaCa
2.944
0.402



MDA-231
1.919
0.316



Lu-134
2.480
0.623



GT3TKB
2.243
0.260



OCUM
2.745
0.308



MC-5
2.268
0.403



H-31
1.957
0.187



H-48
1.960
0.198



PAN-4
1.448
0.277



AZ-521
2.102
0.365










Reference DNA: promega human (male) genomic DNA=2 copies

Mean and standard error from 3 independent experiments.

Example 2
Examination with Formalin-Fixed Paraffin Sections of Human Tumor Strains
The present invention is expected to provide a method for predicting sensitivities to anticancer agents which is widely applicable clinically and yet highly reliable. One of the major reasons for this expectation resides in a point that formalin-fixed paraffin-embedded samples stored in clinical scenes for histopathological examination are believed to be useful as test materials. Then, the present inventors examined whether or not it is possible to detect the copy number of DPD gene by FISH method using paraffin-embedded tissue samples as test materials. As experimental materials, in vivo human tumor strains (Lu-130 and PAN-4) were used. Slide samples (5 μm thick) cut out from paraffin blocks were subjected to deparaffinization and dried. These slide samples were dipped in PBS for 5 min, protease-treated in pepsin/0.1 M HCl at 37° C., washed with PBS sufficiently and then dehydrated and dried with 70% and 100% ethanol.
A probe for detecting DPD gene was prepared as described below. Briefly, a vector (pBeloBAC11; Funakoshi) containing a BAG clone with a DPD gene region (clone ID: CTD-3236P20 (Open Biosystems)) was transfected into Escherichia coli (DH10B; Funakoshi), which was then cultured for expansion. From the resultant cells, BAC DNA was isolated. Cy3-dUTP was allowed to be taken into BAC DNA by nick translation to thereby prepare 1p21 BAC DNA probe. It was confirmed that this probe produces a signal at an appropriate position of the short arm on chromosome 1 using chromosomal samples (Chromosome Science Labo, Ltd.).
This probe was applied to the samples pre-treated as described above. Then, the samples were denatured for 13 min on a hot plate at 90° C., followed by hybridization overnight at 37° C. As a buffer, a solution containing 50% formamide, yeast tRNA, salmon sperm DNA and 2×SSC. Hybridized samples were washed with 50% formaldehyde/2×SSC and 1×SSC, counterstained with DAPI and mounted with an anti-bleaching agent, followed by detection of probe signals. For signal detection and data analysis, Leica CW4000 System (Leica) was used.
Experimental Results
Verification of DNA Probe
When the probe was mapped on chromosome samples which had undergone Hoechst G-banding, the probe was mapped at around 1p21 or 1p22, each of which is an appropriate genomic region for DPD gene. Thus, good signals were detected (FIG. 6).
FISH Analysis with Paraffin-Embedded Sections
FISH images obtained from human tumor strains PAN-4 and Lu 130 are shown in FIGS. 7 and 8. In both samples, clear signals could be obtained. Further, good images with low background were obtained. Out of the counterstained nuclei, 50 or more nuclei which were well isolated on the image were counted. Then, the copy number of DPD gene per cell was determined. In Lu130, 2.3 signals/nucleus were detected. In PAN-4, 1.4 signals/nucleus were detected. These values show a tendency similar to those values obtained in real-time PCR (3.1 and 1.5) (Example 1). Thus, it was shown that the detection results by FISH are reasonable. That is, according to the signal value by FISH method, the DPD gene number in Lu130 was more than 2 copies per cell and therefore this tumor's sensitivity to 5-FU-based anticancer agents was predicted to be low. Likewise, the DPD gene number in PAN-4 was less than 2 copies per cell and therefore this tumor's sensitivity to 5-FU-based anticancer agents was predicted to be high. These predictions and the results of in vivo antitumor effects (Example 1) were consistent in all of the 4 drugs tested in Lu-130; and consistent in 3 drugs out of the 4 drugs tested in PAN-4.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
INDUSTRIAL APPLICABILITY
The present invention has found that the copy number of DPYD gene affects sensitivities to 5-FU-based antitumor agents. With this finding, it has become possible to predict the sensitivity of a subject to 5-FU-based antitumor agents. The prediction result is applicable to cancer therapy.
SEQUENCE LISTING FREE TEXT


    <SEQ ID NO: 1>


SEQ ID NO: 1 shows the DNA sequence of human-derived DPYD.

    <SEQ ID NO: 2>


SEQ ID NO: 2 shows the amino acid sequence of human-derived DPYD.

    <SEQ ID NO: 3>


SEQ ID NO: 3 shows the DNA sequence of mouse-derived DPYD.

    <SEQ ID NO: 4>


SEQ ID NO: 4 shows the amino acid sequence of mouse-derived DPYD.

    <SEQ ID NO: 5>


SEQ ID NO: 5 shows the DNA sequence of the forward primer used to determine the copy number of DPYD gene.

    <SEQ ID NO: 6>


SEQ ID NO: 6 shows the DNA sequence of the reverse primer used to determine the copy number of DPYD gene.

    <SEQ ID NO: 7>


SEQ ID NO: 7 shows the DNA sequence of the forward primer used to determine the copy number of reference genomic sequence LINE-1.

    <SEQ ID NO: 8>


SEQ ID NO: 8 shows the DNA sequence of the reverse primer used to determine the copy number of reference genomic sequence LINE-1.

    <SEQ ID NO: 9>


SEQ ID NO: 9 shows the DNA sequence of the forward primer used to quantitatively determine DPYD mRNA.

    <SEQ ID NO: 10>


SEQ ID NO: 10 shows the DNA sequence of the reverse primer used to quantitatively determine DPYD mRNA.

    <SEQ ID NO: 11>


SEQ ID NO: 11 shows the DNA sequence of the TaqMan probe used to quantitatively determine DPYD mRNA.

    <SEQ ID NO: 12>


SEQ ID NO: 12 shows the DNA sequence of the forward primer used to quantitatively determine ACTB as an internal standard.

    <SEQ ID NO: 13>


SEQ ID NO: 13 shows the DNA sequence of the reverse primer used to quantitatively determine ACTB as an internal standard.

    <SEQ ID NO: 14>


SEQ ID NO: 14 shows the DNA sequence of the TaqMan probe used to quantitatively determine ACTB as an internal standard.

    <SEQ ID NO: 15>


SEQ ID NO: 15 shows the DNA sequence of the forward primer used to quantitatively determine GAPD as an internal standard.

    <SEQ ID NO: 16>


SEQ ID NO: 16 shows the DNA sequence of the reverse primer used to quantitatively determine GAPD as an internal standard.

    <SEQ ID NO: 17>


SEQ ID NO: 17 shows the DNA sequence of the TaqMan probe used to quantitatively determine GAPD as an internal standard.

## Claims

1. A method of predicting the sensitivity of a cancer cell to a 5-fluorouracil-based anticancer agent using the copy number of dihydropyrimidine dehydrogenase gene as an indicator, comprising:
providing a reagent for hybridizing to a region of dihydropyrimidine dehydrogenase gene present in cancer cells but not found in other genomic DNA;
reacting the reagent with dihydropyrimidine dehydrogenase gene in cancer cells derived from a subject; and
generating data based on the reaction with dihydropyrimidine dehydrogenase for correlating the copy number of dihydropyrimidine dehydrogenase gene to the subject's sensitivity to the 5-fluorouracil-based anticancer agent,
wherein the reagents are a pair of oligonucleotide primers represented by SEQ ID NO: 5 and SEQ ID NO: 6.

2. A method of predicting the sensitivity of a cancer cell to a 5-fluorouracil-based anticancer agent, comprising:
providing a reagent for hybridizing to a region of dihydropyrimidine dehydrogenase gene present in cancer cells but not found in other genomic DNA;
reacting the reagent with dihydropyrimidine dehydrogenase gene in cancer cells derived from a subject;
generating data based on the reaction with dihydropyrimidine dehydrogenase for determining the copy number of dihydropyrimidine dehydrogenase gene as an indicator of subject's sensitivity to the 5-fluorouracil-based anticancer agent; and
correlating the determined copy number to the 5-fluorouracil-based anticancer agent based on statistical analysis of the sensitivity of the cancer cells to the 5-fluorouracil-based anticancer agent, wherein the sensitivity is determined to be high for the resultant copy number of 2 or less and to be low for the resultant copy number of more than 2,
wherein the reagent is oligonucleotide primers for amplifying the whole or a part of dihydropyrimidine dehydrogenase gene which the length is about 17 to 25 bases, the primers are configured so that their 3′ end sequences are not complementary to one another to prevent dimerization and so that they do not contain auto-complementary sequences of 4 or more bases to avoid formation of secondary structures within the primers, and
wherein a pair of the oligonucleotide primers is represented by SEQ ID NO: 5 and SEQ ID NO: 6.

3. A method of predicting the sensitivity of a cancer cell to a 5-fluorouracil-based anticancer agent, comprising:
providing a reagent for hybridizing to a region of dihydropyrimidine dehydrogenase gene present in cancer cells but not found in other genomic DNA;
reacting the reagent with dihydropyrimidine dehydrogenase gene in cancer cells derived from a subject;
generating data based on the reaction with dihydropyrimidine dehydrogenase for determining the copy number of dihydropyrimidine dehydrogenase gene as an indicator of subject's sensitivity to the 5-fluorouracil-based anticancer agent;
correlating the determined copy number to the 5-fluorouracil-based anticancer agent based on statistical analysis of the sensitivity of the cancer cells to the 5-fluorouracil-based anticancer agent, wherein the sensitivity is determined to be high for the resultant copy number of 2 or less and to be low for the resultant copy number of more than 2; and
correcting the copy number using data obtained from a reference sequence that varies little in copy number between cancer cells and normal cells,
wherein the reference sequence is represented by a pair of forward primer and reverse primer, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

4. A method of predicting the sensitivity of a cancer cell to a 5-fluorouracil-based anticancer agent, comprising:
providing a reagent for hybridizing to a region of dihydropyrimidine dehydrogenase gene present in cancer cells but not found in other genomic DNA;
reacting the reagent with dihydropyrimidine dehydrogenase gene in cancer cells derived from a subject;
generating data based on the reaction with dihydropyrimidine dehydrogenase for determining the copy number of dihydropyrimidine dehydrogenase gene as an indicator of subject's sensitivity to the 5-fluorouracil-based anticancer agent;
correcting the copy number using data obtained from a reference sequence that varies little in copy number between cancer cells and normal cells; and
correlating the corrected copy number to the 5-fluorouracil-based anticancer agent based on statistical analysis of the sensitivity of the cancer cells to the 5-fluorouracil-based anticancer agent, wherein the sensitivity is determined to be high for the resultant copy number of 2 or less and to be low for the resultant copy number of more than 2,
wherein the reagent is oligonucleotide primers for amplifying the whole or a part of dihydropyrimidine dehydrogenase gene which the length is about 17 to 25 bases, the primers are configured so that their 3′ end sequences are not complementary to one another to prevent dimerization and so that they do not contain auto-complementary sequences of 4 or more bases to avoid formation of secondary structures within the primers, and
wherein a pair of the oligonucleotide primers is represented by SEQ ID NO: 5 and SEQ ID NO: 6.

5. A method of predicting the sensitivity of a cancer cell to a 5-fluorouracil-based anticancer agent, comprising:
providing a reagent for hybridizing to a region of dihydropyrimidine dehydrogenase gene present in cancer cells but not found in other genomic DNA;
reacting the reagent with dihydropyrimidine dehydrogenase gene in cancer cells derived from a subject;
generating data based on the reaction with dihydropyrimidine dehydrogenase for determining the copy number of dihydropyrimidine dehydrogenase gene as an indicator of subject's sensitivity to the 5-fluorouracil-based anticancer agent;
correcting the copy number using data obtained from a reference sequence that varies little in copy number between cancer cells and normal cells; and
correlating the corrected copy number to the 5-fluorouracil-based anticancer agent based on statistical analysis of the sensitivity of the cancer cells to the 5-fluorouracil-based anticancer agent, wherein the sensitivity is determined to be high for the resultant copy number of 2 or less and to be low for the resultant copy number of more than 2,
wherein the reference sequence is represented by a pair of forward primer and reverse primer, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.